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Protocols for Human Immune Monitoring

Flow cytometry and the more recently developed mass cytometry—or cytometry by time-of-flight (CyTOF)— are key technologies broadly used by researchers to study the immune system. Since its invention in the late 1960s, fluorescence-based flow cytometry has advanced considerably and now allows the simultaneous detection of 18-20 proteins expressed by individual cells. Fluorescence-based flow cytometry has enable researchers to better understand the complexity of the immune system. Yet, the phenotypic and functional heterogeneity of immune cells has trigger the need for even more proteins to be detected at once. To overcome the limitation imposed by the spectral overlap of fluorochromes, CyTOF technology uses elemental isotopes instead of fluorochromes to label antibodies. CyTOF enables simultaneous detection of 30-50 proteins and could, in theory, detect up to 100 markers. Using these technologies, immunologists have developed a broad range of staining procedures, gating strategies, and functional assays to better understand the immune system.

Our special issue on human immune monitoring presents a comprehensive collection of detailed and peer reviewed protocols focused on human sample processing for flow cytometry analysis (section 3), immune assays to assess the composition, phenotype and functionality of human immune cells by fluorescence-based flow cytometry (section 2) and by CyTOF technology (section 1). Bio-protocols’ uniquely interactive platform supports communication between scientists – through feedback, Q&A, and protocol updates sections – and will allow you to set up cytometry based technologies for your research. Bio-protocol is a living platform and our Protocols for Human Immune Monitoring special issue will grow with the cytometry field, giving you access to the latest developments.

CyTOF

Efficiency of drug and gene delivery via nonviral vehicles is contingent on proper cellular uptake and intracellular release. Further, various cargos, such as nucleases for gene editing or inhibitors for endosomal receptors, require transport to specific compartments of the cell. Hence, characterization of cellular uptake and endocytic pathways is ...

Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different cell signaling pathways. These pathways include T and B cell signaling, regulating growth and ...

The ability to assess the function of a range of cytokine, antigen receptor, and Toll-like receptor (TLR) signaling pathways in a range of immune cells could provide a kind of fingerprint of the state of the human immune system. The mass cytometry or CyTOF, platform allows for the parallel application of about 40 labeled antibodies to a single ...

Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with ...

In this protocol, we use a CyTOFTM mass cytometry to collect single-cell data on a large number of cytokines/chemokines as well as cell-surface proteins that characterize T cells and other immune cells. The current selected mass window in AW 103-203 includes the lanthanides used for most antibody labeling, along with iridium and rhodium ...

Flow Cytometry

Production of cytokines plays an important role in the immune response. Cytokines are involved in many different pathways including the induction of many anti-viral proteins by IFN gamma, the induction of T cell proliferation by IL-2 and the inhibition of viral gene expression and replication by TNF alpha. Cytokines are not preformed factors but ...

Proliferative capacity and degranulation are important features of antigen-specific CD8+ T cells. By combining tetramer staining with a CFSE staining, we were able to enumerate the total number of antigen-specific T cells, as well as their number of divisions upon antigen-specific stimulation during a week. In addition, we performed ...

This is a general protocol to stain whole human blood for flow analysis with minimal spontaneous activation of monocytes. This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research.